The development of assays for measuring the presence and amount of desired substances is highly desirable for a variety of purposes, including for medical, veterinary, research, and environmental uses. It is further desirable to design and isolate molecules having an activity which is regulatable by a desired substance. Assays can then be designed to detect the amount and presence of a desired substance, such as an analyte in a test sample, utilizing the ability of the analyte to directly or indirectly (e.g., by competition) regulate the molecule""s activity. Assays can then be designed which utilize these regulatable activities.
The present invention relates to a chimeric target molecule having an activity which can be regulated or modulated by a binding molecule. The invention also relates to methods of using the chimeric target molecule to detect the presence and/or amount of a desired analyte in a sample. The analyte is a binding molecule, or a competitor of a binding molecule, which binding molecule, upon binding to the target molecule, alters the activity of the target molecule in a detectable way. In one aspect of the invention, a binding molecule binds to the chimeric molecule, inactivating it. An analyte in a test sample competes and/or displaces the binding molecule from the chimera, reactivating it. The reappearance of activity in the presence of the analyte indicates its existence and amount in the test sample. Another aspect of the invention relates to a binding molecule which regulates a chimeric target molecule and methods of producing it.
In accordance with the present invention, a desired target molecule (TM) can be modified to have at least one binding site moiety (BSM) to which a binding molecule (BM) can attach. Upon attachment of the BM to the BSM, an activity associated with the TM is altered in a detectable way, e.g., increasing or reducing the activity of the TM. Thus, the BSM can act as a regulatory switch, turning on or off (all or in part) an activity of a desired TM in response to the binding of a BM. The BSM can also be selected so that binding of the binding molecule regulates the activation of the target molecule. In accordance with the present invention, a mimetope is the preferred BSM. A BSM can be engineered into a target molecule by the insertion of sequences, by the replacement of sequences present in the molecule with new sequences, by mutagenesis of sequences already present in the molecule, etc. Engineering can be accomplished according to methods available to the skilled worker.
The target molecule can be selected for a desired detectable activity. For example, the TM can be: xcex2-lactamase: P. Soumillion et al., J. Mol. Biol., 237:415:-422, 1994; Plasmin: L. Jespers et al., conference communication; Prostate specific antigen: R. Ecrola et al., Biochem. Biophys. Res. Comm., 200:1346-1352, 1994; Subtilisin: P. Soumillion et al., Appl. Biochem. Biotechnol., 47:175-190, 1994; Trypsin: D. R. Corey et al., Gene, 128:129-134, 1993; Alkaline phosphatase: J. McCafferty et al., Prot. Enging., 4:955-961; xcex2-galactosidase: I. N. Maruyama et al., Proc. Natl. Acad. Sci. USA, 91:8273-8277, 1994; Staphylococcal nuclease: J. Ku and P. G. Schultz, Bioorg. Med. Chem., 2:1413-5, 1994; and J. Light and R. A. Lerner, Bioorg. Med. Chem., 3:955-67, 1995; Glutathione transferase: M. Widersten and B. Mannervick, J. Mol. Biol., 250:115-122, 1995; Lysozyme: K. Maenaka et al., Biochem. Biophys. Res. Comm., 218:682-687, 1996; and Catalytic antibodies: K. D. Janda et al., Proc. Natl. Acad. Sci USA, 91:2532-2536, 1994.
The above-mentioned target molecules have been displayed on phage. They are directly amenable to the method of selection of BSM. Other enzymes can also be displayed on phage and are useful for the present invention, e.g., esterases, pyruvate kinase, glucose oxidase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, luciferase. The TM can also be a protein possessing a fluorescent activity (e.g., green fluorescent protein, GFP: Chalfie et al., 1994, Science, 263:802; Cheng et al., 1996, Nature Biotechnology, 14:606; Levy et al., 1996, Nature Biotechnology, 14:610) which is modulated by binding of a BM to a BSM contained within the fluorescent protein. The TM can also be a regulatory molecule which activates/inactivates a second molecule having a detectable activity. For instance, a GTPase activating protein (GAP) stimulates a G-protein, such as ras. The ability of a GAP to activate a G-protein can be modulated by engineering a BSM into the GAP. Upon attachment of a BM to the BSM of a modified GAP, the stimulating activity of the GAP can be modulated. Its upstream effect on G-proteins can be monitored, e.g., by measuring a GTPase activity of the G-protein. See, e.g., Trahey and McCormick, Science, 238:542-545, 1987. The TM can also be a subunit of another protein which itself possesses enzymatic or another detectable activity. Additionally, the TM can be a nucleic acid enzyme, e.g., a ribozyme, a hammerhead enzyme, RNAse P, or a hairpin enzyme. If a nucleic acid is used as the target molecule, the engineered binding site moiety would usually comprise nucleotides, either modified or naturally-occurring. The TM can also be transcription activators and repressors regulated in in vitro transcription and translation systems; detection of activity can be accomplished at the level of the activity of the expressed enzyme or fluorescent molecule.
The activation of a chimeric molecule can also be regulated by a BM. The simplest example of activation is the proteolytic cleavage of a peptide bond in a zymogen to transform it into an enzyme. A classical example is the activation of a serine protease, or more specifically the activation of chymotrypsinogen into chymotrypsin by proteolytic cleavage of the peptide bond Arg15-Ile16 by trypsin. An antibody binding to an epitope or a mimotope engineered in the region of the cleaved peptide bond can inhibit the activation. Another example is the inhibition of the phosphorylation or dephosporylation of an enzyme whose activity is regulated by its state of phosphorylation. Glycogen phosphorylase is an example: when it is phosphorylated on Ser14, it is essentially in its active form, dephosphorylation deactivates the enzyme. Binding of an antibody to a engineered epitope or mimotope in the vicinity of the phosphorylation site would interfere with the activation/deactivation mechanism by phosphorylase kinase and phosphoprotein phosphatase respectively.
More generally any postraductional modification of an enzyme, that contributes to modulate its activity, can be interfered with by binding a foreign molecule to a BSM (e.g., an antibody).
The term xe2x80x9cchimericxe2x80x9d target molecule, e.g., a xe2x80x9cchimeric enzyme,xe2x80x9d means the resultant product after the binding site moiety has been inserted into the target molecule or after a portion of the target molecule has been replaced by the binding site moiety. For clarity, before engineering of the BSM, the target molecule is referred to as the starting target molecule. Thus, if an enzyme is the starting material, it is referred to as the xe2x80x9cstarting enzyme.xe2x80x9d After engineering of the BSM, the starting enzyme is identified as a xe2x80x9cchimeric enzyme.xe2x80x9d In the examples below, xcex2-lactamase is used as a starting enzyme into which a binding site moiety comprising amino acids, is engineered to produce a chimeric enzyme. It is chimeric because it is comprised of amino acids of the starting enzyme and amino acids of a binding site moiety.
Target and chimeric molecules can be prepared by methods which are available in the art. For example, genetic engineering can be employed to prepare target and chimeric molecules which comprise amino acid or nucleotide residues. In one embodiment, a cloned gene is employed as the starting material for the starting target molecule and resultant chimeric target molecule. In the examples described below, the cloned gene for the starting enzyme xcex2-lactamase serves as the beginning material to produce the chimeric enzyme. The BSM can be engineered into the starting TM using the various methods available to the skilled worker, e.g., Kunkel, Proc. Natl. Acad. Sci., 82:488-492, 1985; Brennan et al., Proc. Natl. Acad. Sci., 92:5783-5787, 1995. Engineering can also be accomplished using a replacement vector via homologous recombination. For the purposes of the present invention, when a sequence within a starting gene has been mutagenized to the extent that the amino acid sequence differs from the starting sequence, the polypeptide coded for the resultant gene is chimeric. It is chimeric since a different amino acid sequence, i.e., a binding site moiety, has been engineered into the starting target molecule. In the specific example where the starting material is an enzyme, and the enzyme is mutagenized by changing its nucleotide sequence, a resultant chimeric enzyme will comprise an amino acid binding site moiety which has replaced the naturally-occurring amino acid sequences. In one embodiment, the sequence of the gene encoding a wild type enzyme (or other polypeptide) is modified by the site directed mutagenesis according to the Kunkel or Eckstein protocols to introduce two restriction sites upstream and downstream from the region of the gene targeted for engineering; preferentially, a mutation is introduced in the coding sequence at the same time so that the encoded enzyme is inactive; the plasmid, phagemid or phage containing the modified gene will be called the xe2x80x9cvector.xe2x80x9d This vector is digested at the new restriction sites with the corresponding restriction enzymes and the small fragment encoding the sequence between the sites is discarded. In parallel, synthetic degenerate oligonucleotide libraries are prepared; they contain, in between the adequate restriction sites, degenerate nucleotide sequences encoding random replacements of the corresponding residues in the protein sequence. Alternatively, the wild type sequence is replaced by a longer nucleotide sequence that will encode the insertion of a random polypeptide in the corresponding position in the protein sequence. After restriction, the synthetic oligonucleotides are ligated with the purified large fragment of the digested vector and the ligation mixture is used to transform E. coli cells. Typically, libraries containing about 106 and 108 transformants are produced. Clones producing active enzymes are selected from these (see below). Recombination of clones producing active enzymes in two libraries where random mutations are introduced in different parts of the sequence is done to produce enzymes with discontinuous mimotopes.
The invention also relates to nucleic acids which code for a chimeric target molecule. Such a nucleic acid can further comprise various sequences, e.g., an expression control sequence(s) operably linked to a nucleotide sequence coding for the chimeric target molecule. The phrase xe2x80x9cexpression control sequencexe2x80x9d means a nucleic acid sequence which regulates expression of a nucleic acid to which it is operably linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc. An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5xe2x80x2 to a coding sequence, expression of the coding sequence is driven by the promoter. A nucleic acid coding for a chimeric also includes nucleic acids which hybridize to it, e.g., under stringent conditions, such as conditions that allow the selection of at least 95%, 99% nucleotide identity. For a chimeric TM which is a polypeptide, a nucleic acid coding for it includes, e.g., nucleotide degeneracy. Nucleic acids include DNA and RNA.
Chemical and/or synthetic methods can also be used to create the chimeric molecule, e.g., the methods of building compounds by combinatorial chemistry, as the skilled worker would know.
After modification of the starting target molecule to produce a resultant chimeric target molecule, it is desirable to select those chimeric molecules which have retained an activity of the starting target molecule. By the phrase, xe2x80x9cthe chimeric target molecule has an activity of the starting target molecule,xe2x80x9d it is meant that the starting TM has an activity and the resultant chimeric TM has an activity, as well. The activity of the chimeric TM can be different quantitatively or qualitatively from the starting TM. By way of illustration, in the examples below, the starting enzyme is xcex2-lactamase. xcex2-lactamase is an enzyme which hydrolyzes a xcex2-lactam bond. Various compounds can be used as substrates, including penicillins, cephalosporins, ampicillin, etc. The activity of the starting xcex2-lactamase is hydrolysis of a xcex2-lactam bond. A chimeric xcex2-lactamase having a binding site moiety, either replacing or inserted in addition to naturally-occurring amino acids, will possess the ability to hydrolyse a xcex2-lactam bind. This activity in the chimeric xcex2-lactamase can be, e.g., greater or less than the starting enzyme (e.g., having a different Kcat), and/or have a different substrate specificity.
After modification by the engineering, e.g., insertion or replacement, of a BSM into the target molecule, the selection of the resultant molecule can be accomplished by various methods as the skilled worker would know. In one embodiment where genetic engineering is utilized, a gene coding for a target molecule, e.g., an enzyme, can be cloned into an expression vector suited for expression of a polypeptide in a desired host. Various hosts are contemplated, including, mammalian cells (e.g., human, monkey, or rodent, such as HeLa, COS, Ltk-, or CHO), insect cells (e.g., Sf9 or Drosophila), bacteria (e.g., E. coli, Streptococcus, or bacillus), yeast, fungi, or plants. See, also Methods in Enzymology, Volume 185, ed., D.V. Goeddel. Sf9 expression can be accomplished in analogy to Graziani et al., Oncogene, 7:229-235, 1992. Filamentous phage systems have been used to express and select peptides in bacteria that attach to binding molecules, including antibodies (Scott and Smith, 249:386-390, 1990; Grihalde et al., Gene, 166:185-195, 1995), streptavidin (Kay et al., 1993; Devlin et al., Science, 249:404-406, 1990), ribonuclease (Smith et al., Science, 228:1315-1317, 1985) and DNA (Rbar and Pabo, 1994). See, also, Jespers et al., Biotechnology, 13:378-382, 1995. See, also, Smith, Science, 228:1315-1317, 1985; Parmley and Smith, Gene, 76:305-318, 1985; de la Cruz et al., J. Biol. Chem., 263:4318-4322, 1988; Bass et al., Proteins, 8:309-314, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382, 1990; Devlin et al., Science, 249:404-406, 1990; McCafferty et al., Nature, 348:552-554, 1990; Clackson et al., Nature, 352:624-628, 1991; Lowman et al., Biochemistry, 30:10823-10838, 1991; J. McCafferty et al., Port. Engng, pp. 955-961, 1991; Kang et al., Proc. Natl. Acad. Sci. USA, 88:4363-4366, 1991; Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982, 1991; Roberts et al., Proc. Natl. Acad. Sci. USA, 89:2429-2433, 1992. Preferred polypeptides for filamentous phage expression systems are those which are properly folded on the phage, or at least, displayed on the phage in a fully active form. To identify whether a desired starting molecule is suitable, a nucleic acid coding for the molecule is cloned into the phage in a manner suitable for expression. The expressed molecule is then assayed for an activity in accordance with conventional methods. Engineering of a BSM into the starting molecule can then be accomplished in accordance with the above-mentioned procedures. See, e.g., Grihalde et al. Expression control sequences are selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression, etc. Other sequences which can be employed, include enhancers such as from SV40, CMV, inducible promoters, or other elements which allow selective or specific cell expression.
A binding molecule can bind to a specific portion of a macromolecule called an epitope or a determinant. The epitope can be a linear determinant or a conformational determinant. See, e.g., Abbas et al., Cellular and Molecular Immunology, Second Edition, W.B. Saunders Co., 1991, especially, pages 47-49. A xe2x80x9cmimetopexe2x80x9d is a determinant which is recognized by the same binding molecule as a particular xe2x80x9cepitopexe2x80x9d but which has a different composition from the xe2x80x9cepitope.xe2x80x9d For example, a binding molecule can be an antibody which recognizes (i.e., binds to) an epitope comprising a linear sequence of amino acids. A xe2x80x9cmimetopexe2x80x9d of this epitope comprises a different linear sequence of amino acids but which is still recognized by the same antibody. The xe2x80x9cmimetopexe2x80x9d differs by at least one amino acid from the xe2x80x9cepitope.xe2x80x9d A mimetope can mime a hapten and other molecules, including nonproteinaceous molecules or moieties, e.g., carbohydrate, biotin, etc. As mentioned, the mimetope can also be a conformational determinant formed by amino acid residues or other constituents from separated portions of the chimeric molecule. Further, the mimetope can comprise constituents (e.g., amino acids) already present in the starting TM and which remained (i.e., were not replaced) in the chimeric TM. A mimetope can be selected as discussed above and below, e.g., in the examples, by engineering random amino acids into a target and screening for recognition by a desired binding molecule.
An advantage of employing a mimetope is that no knowledge of the structure of the epitope is required. This knowledge is in general difficult to acquire, particularly if the epitope is non-linear. In one aspect of the invention, a library of mimetopes is created and engineered, e.g., inserted, into a target molecule, preferably into a loop. The resultant chimeric molecule is then screened or selected for retention of activity. The mimetope can be a random sequence, e.g., containing five amino acids, preferably six amino acids (a random hexapeptide), or seven, eight, nine, ten, amino acids in length. In this aspect of the invention, upon identification of chimeric target molecules which have retained activity, they are then screened for recognition by the desired binding molecule. The binding molecule can be an antibody to a carbohydrate or other non-proteinaceous hapten or non-hapten, or an amino acid sequence. In especially the latter case, no sequence information is required to implement the invention.
The binding site moiety can be engineering into any desired position in the target molecule, including as a fusion with the N- and C-termini. One or more, e.g., 2, 3, 4, or 5, BSMs can be engineered into the target moiety at adjacent or different regions. Multiple engineering, e.g., insertions or replacements, to the target molecule can be made for a variety of reasons, e.g., to contribute to the mimetope (e.g., the mimetope can be comprised of amino acids contributed by engineering at two different sites in the target molecule), to provide more than one site to which a binding molecule can bind, to provide one site at which a BM activates the enzyme and another site at which a second BM inactivates an enzyme, etc. An advantage of inserting or replacing amino acid sequences with a mimetope at two sites (or more) is that a discontinuous mimetope can be constructed, providing for high affinity sites to which a binding molecule can attach. Preferably, as discussed above, the resultant chimeric TM retains at least some of its activity after engineering of the BSM. In addition, attachment of a BM to the BSM results in regulating the aforementioned activity of the chimeric target molecule. The latter two aspects, retention of an activity and regulation of the retained activity of the resultant chimeric molecule by a binding molecule, are preferred aspects of the invention. Thus, a preferred site of engineering, e.g., insertion, is a position where the activity of the TM is not eliminated but which, when replaced or modified by the addition of amino acid residues, can act as a regulatory switch for TM activity. By the phrase xe2x80x9cwhereby the activity of the chimeric target molecule is modulated upon binding of a binding molecule,xe2x80x9d it meant that attachment of the binding molecule to the chimeric TM, preferably at the BSM, affects the activity of the chimeric TM in a detectable way. If the chimeric TM is an enzyme such as xcex2-lactamase, the binding molecule will affect its activity in hydrolyzing the xcex2-lactam bond. The affect of the binding molecule can be to reduce or even eliminate the activity, e.g., reduce or eliminate its ability to cleave the xcex2-lactam bind. The binding molecule can also affect activity in other ways, e.g, activate it, increase it, change its specificity, activate it, etc.
Binding of the BM to the chimeric molecule, preferably at the BSM, can affect activity in various ways. For example, a chimeric TM can exist in at least two conformations, an active and inactive conformation. At equilibrium, a population of chimeric TMs will contain a mixture of molecules, some in the active and some in the inactive conformation. A BM can be selected that binds to an inactive conformation of a TM. When added to the chimeric TM population, attachment of the BM to the inactive TMs can shift the equilibrium of the mixture to the inactive conformation. As a consequence, the mixture will have less activity in the presence of the BM than in its absence. Thus, the binding molecule modulates the activity of the chimeric TM by shifting the population of chimeric TMs to an inactive conformation, thereby reducing the population""s activity as a whole.
The binding molecule can inactivate the activity of the chimeric TM. By the term xe2x80x9cinactivate,xe2x80x9d it is meant that the activity of the chimeric TM is reduced or weakened. The binding molecule can inactivate the chimeric TM completely so that it possesses no, or only negligible, activity, or it can inactivate only part of its activity, e.g., where the Kcat is reduced. Attachment of the binding molecule to an inactive conformation of the chimeric TM is an example where the binding molecule inactivates the activity of the chimeric TM. A selected starting enzyme can be serine protease that can exist in two different conformations: an active and an inactive one. The inactive conformation is similar to that of the corresponding zymogen. The equilibrium can be shifted from the active into the inactive conformation by disrupting the salt bridge maintaining the enzyme in its active conformation; this can be done by a pH increase leading to deprotonation of the amino terminal of the peptide chain involved in the salt bridge or by chemical modification of this amino terminal. The energetics of the salt bridge are such that the active conformation is not strongly stabilized (2.9 Kcal/mol, see: A. R. Fersht, J. Mol. Biol., 64:497-509, 1972) so that the equilibrium can be relatively easily shifted to the inactive form. Binding of a monoclonal antibody to the amino acid terminal can shift the equilibrium by several orders of magnitude.
The site where a BSM is engineered, e.g., inserted into and/or replaced, in the TM can be selected by various ways as the skilled worker would know. For example, if the three-dimensional (3D) structure of the TM is known, a site can be selected by specifically identifying a desired location on the molecule to engineer. For some purposes, it may be desirable to select an exposed site on the surface of the target molecule, where the site is available for attachment by the binding molecule. 3D-structure can be determined according to empirical means, e.g., by crystallography, and/or, it can be deduced from known structures and amino acid sequence data. See, e.g., Holm and Sander, Science, 273:595-602, 1995. If the 3D-structure is not known, the site of engineering can be selected on the basis of other information, e.g., when the structure of the protein is not known, sites susceptible to limited proteolysis or sites strongly predicted to be loops by secondary structure prediction or by analysis of hydrophobic patterns are suitable for engineering, e.g., insertion or replacement. Alternatively, a BSM can be engineered at random positions within the TM.
The engineered site is preferably not at the active site, more preferably it is at a location remote from it, e.g., about 1, 5, 15, 20, or 25 xc3x85 from it. The activity must be regulatable by binding to the inserted or replaced sequence, irrespective of whether the modification is close or remote from the active site.
As mentioned above, an aspect of the present invention involves chimeric target molecules which have an activity that can be regulated or modulated by a binding molecule. In one preferred embodiment, random peptide sequences are engineered at a selected site on a target molecule, e.g., an enzyme. The first step is to select resultant chimeric molecules which retain the desired activity. If an enzyme activity is the desired activity, then a selection assay can be designed for it. Selection can be accomplished by color (e.g., where cleavage by the enzyme produces an end-product having a detectable color), by conferring resistance to clones expressing an active enzyme (e.g., drug resistance), etc. In one embodiment, screening is performed by plating a library on solid medium, adding a chromogenic or fluorogenic substrate, and observing product development in individual colonies. In vivo selection can be applied when the molecule is necessary for growth in the presence of antibiotic (antibiotic resistance; this technique is used with the beta-lactamase in the examples), or when the activity is used for complementation of an missing essential gene in auxotrophic bacteria (e.g., auxotrophy for an amino acid). In vitro selection can also be used when the enzyme is displayed on phage; e.g., WO 93/11242.
To measure the activity of the selected enzymes, any classical spectrophotometric, fluorimetric, potentiometric (pHstat) technique can be used. In the particular, the ORIGEN technology can be used for detection of product formation (JACS, 118, 9198-99). A next step of selection is to identify clones which bind to the binding molecule. Selection can be accomplished by antibody panning technique, column chromatography, etc. See, e.g., Grihalde et al., Gene, 166:187-195 (1995); McNally et al., J. Bio. Chem., 270:19744-19751, 1995; O""Neil and Hoess, Curr. Opin. Struct. Biol., 5:443-449, 1995. In one embodiment, the chimeric target molecule is expressed on the surface on the host cell (e.g., a bacteria, a insect cell, a mammalian cell) and selection can be accomplished without cell lysis. The chimeric target can also be expressed within the host cell and selection accomplished after, e.g., permeabilizing or lysing the cells, or otherwise making the expressed product accessible to the binding molecule.
A binding molecule means a molecule that specifically binds or attaches to a binding site moiety. By the term xe2x80x9cspecific,xe2x80x9d it is meant that the binding molecule recognizes the defined sequence of amino acids within or including the amino acid sequence of the binding site moiety. Specificity can be a function of the linear amino acid sequence of the binding site moiety, alone, or in combination with amino acids originally present in the target molecule or at an insertion or replacement at another site. Various binding molecules can be employed, including antibodies, polypeptides, aptamers, nucleic acids, drugs, and chemical ligands. Antibodies can be monoclonal, polyclonal, single-chain, genetically-engineered antibodies, etc., as known in the art. See, e.g., Reiter et al., Nature Biotechnology, 14:1239-1245, 1996; Bird et al., Science, 242:423-426, 1988.
A chimeric target molecule can be used to detect the presence or amount of an analyte in test sample. In one embodiment, a chimeric TM is a chimeric enzyme. The chimeric enzyme is contacted with a (1) test sample containing an analyte, and (2) a substrate upon which the chimeric TM enzyme catalytically acts, to form a reaction mixture. The amount of analyte present in the reaction mixture is determined by monitoring or detecting the amount of catalysis of the substrate achieved by the chimeric enzyme, wherein the analyte modulates the catalysis by the chimeric enzyme. A test sample can be any sample containing an analyte whose presence or amount it is desired to be known, e.g., body fluids such as blood, serum, urine, feces, or lymph, tissue homogenates, biopsies, organ fluids, tissue culture medium, etc. By xe2x80x9canalyte,xe2x80x9d it is meant a molecule whose presence in a test sample is being detected. In one embodiment, the analyte is an antibody, such as an antibody specific for prostate specific antigen (PSA), carcinoma embryonic antigen (CEA), c-erbB2, products of oncogenes, viral (HIV or hepatitis), bacterial (staphylcoccal), and the chimeric TM is a chimeric enzyme. Alternatively, the analyte can be a polypeptide such as any of the aforementioned proteins or fragments thereof. Binding or attachment of the antibody to the chimeric enzyme can modulate catalysis of the substrate by the chimeric enzyme. Modulation of activity is discussed above. In a preferred example, the enzyme activity of the chimeric enzyme is reduced (inactivated) by the antibody. Thus, the presence of the analyte antibody in the test sample can be determined by monitoring or detecting the reduction of activity manifested by the chimeric enzyme, either as individual molecules or as a population. Alternatively, the analyte is a polypeptide. When the chimeric molecule is combined with an appropriate binding molecule, its activity is modulated. Addition of the analyte, competes and/or displaces the binding molecule, reversing its modulatory effect on the detectable activity. The enzyme assay can be performed in accordance with known procedures. For example, the activity can be monitored temporally, kinetically, or by end-point. The chimeric enzyme can be in solution or on a solid support, e.g., directly coupled or via biotin-strepavidin coupling, to materials such cellulose, Sephadex, plastics, polypropylene, polystyrene, polyvinyl, cellulose nitrate, polythylene, nylon, polymethylmetaacrylic, etc. The coupling can be accomplished as one having skill in the art would know. See, e.g., Methods in Enzymology, Volume 73, for various techniques on substrates, coupling, and assays in general. By the term xe2x80x9ccontactingxe2x80x9d the chimeric molecule with a test sample containing analyte or binding molecule, it is meant that the analyte or binding molecule is brought into contact with the chimeric molecule by a desired means. The contact can be accomplished by: adding a test sample to a solution containing the chimeric TM, dipping a solid support containing the chimeric enzyme into a solution containing the analyte or BM, dropping a solution containing an analyte on to a solid support containing the chimeric TM, etc. If a substrate is used, e.g., where a chimeric TM is an enzyme, the substrate can be contacted with the chimeric enzyme at the same time as the analyte, or before or after, i.e., simultaneously or sequentially.
As mentioned, the chimeric TM can be any molecule having a desired activity, e.g., enzymatic, fluorescent, activating, complementary, etc. Assays for detecting an analyte can be tailored as one of ordinary skill in the art would know for monitoring or detecting the change in activity of the selected chimeric TM.
In another aspect of the present invention, the activity of a reaction mixture, comprising a chimeric enzyme and an analyte (a first binding molecule) which modulates the activity of the chimeric enzyme, can be further affected by a second binding molecule. The second binding molecule can act as a direct competitor of the analyte, competing for the same site as the analyte. In one embodiment, the analyte inactivates the activity of the chimeric TM. The second binding molecule acts as antagonist of the analyte, competing for the same site of the chimeric TM but ineffective in inactivating it. Consequently, addition of the second binding molecule will result in the restoration of activity in the reaction mixture. The second binding molecule can also antagonize the action of the analyte by inactivating the analyte, itself, without site-specific competition. In this embodiment, the second binding molecule can, e.g., be an antibody which prevents the analyte from attaching to the chimeric TM and thus reduces the analytes ability to inactivate the chimeric TM. The second binding molecule can be prepared in the same way as described above for the binding molecule.
The assays of the present invention are useful for medical, veterinary, environmental, and various diagnostic uses, e.g., for detecting diseases, pathogenic disorders, environmental contamination, tissue culture contamination, etc. For example: the presence of cancer in a patient can be determined by detecting the presence of a characteristic antigen or antibody. It is known that individuals with cancer can have elevated levels of various antigens, such as prostate-specific antigen (PSA) or carcinoma embryonic antigen (CEA).
In another aspect of the present invention, an analyte is a competitor of a binding molecule. The presence or amount of competition with the binding molecule is used to ascertain its presence. An example of such a process is described in mimetope recognized by a antibody specific for a desired molecule is prepared (in the example, it is prostate-specific antigen or xe2x80x9cPSAxe2x80x9d). Binding of the antibody to the mimetope reduces the activity of the chimeric molecule. The analyte (in the example, it is PSA) competes with the antibody for binding to the mimetope. Thus, if analyte is present, less of the antibody binds to the chimeric molecule. With less antibody bound to the chimeric molecule, the chimeric molecule is more active than in the absence of the analyte. This is illustrated in Example 3.